The separation of DNA fragments by polyacrylamide or agarose gel electrophoresis is a well-established and widely used tool in molecular biology (Sharp, P. A. et al., "Detection of two restriction endonucleases activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis," (1973) Biochemistry 12:3055). The standard technique for viewing the positions of the separated fragments in a gel involves the use of an ultra-violet (UV) transilluminator (Brunk, C. F. and Simpson, L., "Comparison of various ultraviolet sources forfluorescent detection of ethidium bromide-DNA complexes in polyacrylamide gels," (1977) Analytical Biochemistry 82:455). This procedure involves first staining the gel with a fluorescent dye such as ethidium bromide or SYBR.RTM. Green I. The DNA fragments, which bind the dye, are then visualized by placing the gel on a light-box equipped with a UV light-source. Typically the UV source, in combination with a built-in filter, provides light with an excitation maximum of around 254, 300 or 360 nm. The UV light causes the DNA-bound dye to fluoresce in the red (ethidium bromide) or green (SYBR Green I) regions of the visible light spectrum. The colored fluorescence allows visualization and localization of the DNA fragments in the gel. The visualization of DNA in a gel is used either to assess the success of a gene cloning reaction as judged by the size and number of DNA fragments present, or to identify a particular sized fragment which can be cut out from the gel and used in further reaction steps.
Transilluminators used in the art to visualize fluorophors are described in a number of patents, including U.S. Pat. Nos. 5,347,342, 5,387,801, 5,327,195, 4,657,655, and 4,071,883. Clinical examination of skin anomalies causing fluorescence have been described in U.S. Pat. No. 5,363,854 using visible light images as a control.
The use of UV light for viewing molecules in gels has two major disadvantages: (1) It is dangerous. The eyes are very sensitive to UV light and it is an absolute necessity that the viewer wear eye-protection, even for brief viewing periods, to prevent the possibility of serious damage. More prolonged exposure to UV light results in damage to the skin tissues (sunburn) and care must be taken to minimize skin exposure by wearing gloves, long-sleeved jackets and a full-face mask. (2) DNA samples are damaged by exposure to UV light. It has recently been documented by Epicentre Technologies that a 10-20 second exposure to 305 nm UV light on a transilluminator is sufficient to cause extensive damage to the DNA. This period of time is the absolute minimum required to excise a DNA band from a gel.
An alternative to UV transillumination involves the use of laser light sources. However, the use of laser light is not applicable to the simple and direct viewing of a DNA gel by the human eye. The extremely small cross-section of the laser light beam requires that a typical DNA gel be scanned by the laser, the fluorescence intensity at each point measured electronically and stored digitally before a composite picture of the DNA gel is assembled for viewing using computer software.
Visible light boxes for artists' uses are known to the art for visualizing non-fluorescing materials, e.g., as described in U.S. Pat. No. 3,802,102. The use of visible light to detect certain fluorescent dyes is suggested, e.g., in Lightools Research web page. However, no enabling disclosure for making such devices is provided. None of these references provides devices or systems for viewing fluorescence patterns using visible light.
Despite the recent development of dyes fluorescing in the visible spectrum (Haugland, R. [1996] "Handbook of Fluorescent Probes and Research Chemicals, Sixth Edition," Molecular Probes, Inc., Eugene, OR, pp. 13-18, 25-29, 29-35), transilluminators and other devices to take advantage of the properties of such dyes have not been made available to the public. It is an object of this invention to provide devices and methods for directly and indirectly viewing and measuring patterns of fluorescence not involving the use of UV transillumination but rather being capable of using sources of visible light such as ordinary lamps, as opposed to lasers and the focused lights used in standard fluorometers.
All publications referred to herein are incorporated by reference.